PITUITARY RECEPTORS FOR GnRH AND ESTRADIOL, AND PITUITARY CONTENT OF GONADOTROPINS IN BEEF COWS.
I. CHANGES DURING THE ESTROUS CYCLE 1
T.M. Nett, D. Cermak, T. Braden, J. Manns 2 and G. Niswender
Department of Physiology Colorado State University
Fort Collins, Colorado 80523
Received November 11, 1986
ABSTRACT
To further characterize the endocrinological changes in the hypothalamo-hypophyseal axis thoughout the bovine estrous cycle, cycling beef heifers (n = 24) were randomly assigned to six groups. These heifers were slaughtered 6, 12, 18, 19, 20 or 21 days following their previous estrus (day 0). Anterior pituitaries and hypothalami were collected. Hypothalami were divided into the preoptic area and medial l~asal hypo- thalamus, and content of gonadotropin-releasing hormone (GnRH) was quantified by radioimmunoassay. Contents of luteinizing hormone (LH) and follicle stimulating hor- mone (FSH) in the anterior pituitary gland were quantified by radioimmunoassay. Membrane receptors for GnRH were quantified by a standard curve technique and receptors for estradiol in anterior pituitary cytosol were quantified by saturation analysis. There was no significant change in content of GnRH in the hypothalamus or content of FSH in the anterior pituitary on any of the days examined; however, content of GnRH in the preoptic area was lower (P<.I) on day 19 postestrus. Cytosolic receptors for estradiol increased (P<.05) on day 18 post-estrus and returned to baseline by day 19. Content of LH and the number of receptors for GnRH in the anterior pituitary gland decreased (P<.01) on day 19 postestrus, and the number of receptors for GnRH remained low through day 21 postestrus. The reduction in anterior pituitary content of LH was transient indicating that synthesis of LH restores pituitary content to preovulatory levels before the number of receptors for GnRH returns to normal.
INTRODUCTION
The re la t ionship be t ween serum concent ra t ions of gonado t rop ic ho rmones and ovarian func t ion dur ing the bovine estrous cycle has been descr ibed p rev ious ly (1 ,2) ; however , regula t ion of the hypo tha lamo-hypophysea l axis is less clear. Pi tui tary conten t o f gonadot rop ins (3) , and sensit ivi ty of the pi tui tary to exogenous gonadot ropin- re leas ing ho rmone (GnRH; 4,5) dur ing the bovine estrous cycle have been examined , but cor re la t ion of these factors and others such as hypo tha l amic con ten t o f GnRH and pi tu i tary recep to rs for GnRH and estradiol , par t icu lar ly a round the t ime of the luteinizing ho rmone (LH) surge, has been lacking.
Pi tui tary concen t ra t ion of recep tors for GnRH has been r epor t ed to increase on the day of proes t rus in rats (6-8) and hamsters (9) as wel l as dur ing the pe r iovu la to ry pe r iod of the ewe (10) . The cur ren t s tudy was under taken to quant i fy hypo tha l amic conten t of GnRH, pi tu i tary conten t of LH and foll icle- s t imula t ing h o r m o n e (FSH) and to quant i fy the n u m b e r of hypophysea l re- cep tors for GnRH and estradiol at different stages of the bovine estrous cycle wi th emphas is g iven to the pe r iod a round the p reovu la to ry surge of LH and ovulat ion.
Preparat ion o f Tissue. Normally cycl ing crossbred beef heifers were ran- domly assigned to be s laughtered 6, 12, 18,19, 20 or 21 days ( four /g roup) fo l lowing the previous estrus. Animals were t ransported to a local abbatoir on the morn ing they were to be killed. Pituitaries and hypothalami were co l lec ted wi th in 15 min of slaughter. Pituitaries were immediate ly p laced in ice cold TED buffer (10% glycerol , 10 mM Tris, 1.5 mM EDTA, 1 mM dithiothrei tol ; pH 7.6) . Hypothalami were immedia te ly divided into the p reop t ic area (POA) and medial basal hypothalamus (HYP) as descr ibed previously (11) and frozen on dry ice. The median eminence was des t royed in approximate ly half the animals by the captive bol t gun used to kill the heifers. Therefore , data from the median eminence was exc luded from all animals. Each pi tui tary was t r immed of connec t ive tissue, the poster ior lobe was r emoved and the anterior lobe was b lo t ted dry. Anterior pituitaries were homogen ized individual ly in TED buffer at 4C, d i lu ted to a concent ra t ion of 200 mg t issue/ml of buffer and cent r i fuged at 30 ,000 X g for 1 hr. The supernatant was removed and an al iquot was taken for analysis of soluble receptors for estradiol. The remainder was s tored at - -70C unti l analyzed for content of gonadotropins . The pe l le t was resuspended in Tris-HC1 buffer (10 mM Tris, 1 mM CaCI2, .1% BSA, pH 7.4) at a concent ra t ion of 400 mg t i ssue/ml buffer and stored at - 7 0 C for analysis of receptors for gonadotropin-releasing hormone.
S o l u b l e R e c e p t o r s f o r E s t r a d i o l . To quant i fy u n o c c u p i e d solubi l ized re- ceptors for estradiol, all incubat ions were carr ied out at 4C (12) . From a pool of fresh bovine pi tui tary cytosol ic extracts, r ecep to r affinity and capacity were de te rmined by Scatchard analysis. Forty mill igram fresh tissue equivalents (200 ~tl) were added to t r ipl icate tubes containing 100 ~tl of 3H-estradiol ranging in concent ra t ion from .033 to 3.3 nM wi th or wi thou t a lO0-fold excess of estradiol to cor rec t for nonspecif ic binding. After incubat ion for 20 hr free ligand was separated from bound by the addit ion of 250 ~tl of .5% charcoal, .05% dextran, incubat ion for 10 min at 4C fo l lowed by centr i fugat ion (5000 X g) for 5 min.
Specificity of the r ecep to r was assessed by de te rmining the ability of several steroids (1 nM to 100 p.M) to inhibit the binding of 3H-estradiol (1 nM) to 40 mg fresh tissue equivalents f rom a pool of fresh bovine pituitaries. Incubat ion and separat ion were carr ied out as descr ibed above.
Saturation analysis was used to quant i fy estradiol receptors in individual pituitaries. Twenty and 40 mg fresh tissue equivalents (200 ~tl) were incubated as descr ibed above wi th 5 nM 3H-estradiol (100 ~tl). To cor rec t for nonspecific binding, excess estradiol (1 ~tM) was incubated wi th half the tubes. Following incubat ion, separat ion was carr ied out as descr ibed above.
Membrane Receptors f o r GnRH. The ligand used was monoiodo-D-Ala 6- des Gly NH2~0QW-GnRH-ethylamide ([~25I]-D-AIa6-GnRH-EA) p repared by the glucose oxidase- lactoperoxidase t echn ique descr ibed by Wagner et al (13) .
From a pool of f rozen bovine pi tui tary homogenates , rates of association and dissociat ion were de te rmined by adding 2.5 mg tissue (50 ~tl) to tubes con- ta in ing . 175 nM [~2~]-D-AIa6-GnRH-EA (50 ~tl) wi th or wi thou t a 100-fold excess of D-Ala6-GnRH-EA (50 ~I). Total incubat ion vo lume was 150 I.tl.
Specificity of this r ecep to r assay was assessed by de termining the ability of several agonists and antagonists of GnRH to inhibit the binding of [t25I]-D-Ala 6- GnRH-EA to the bovine pi tui tary homogenates . Various concentra t ions of the
HYPOTHALAMO-HYPOPHYSEAL AXIS IN BEEF COWS 125
analogs (.1 nM to .1 mM) were incubated with 2.5 mg of bovine pituitary homogenate and .175 nM [~2q]-D-AIa6-GnRH-EA in a total volume of 150 ~tl and then separated as described previously.
Receptors for GnRH were quantified by a standard curve technique described previously (14). Four and 8 mg of homogenate were incubated at 4C with .175 nM [a25I] D-AIa6-GnRH-EA with or without a 100-fold excess of D-AIa 6- GnRH-EA in a total volume of 150 ~tl for 12 hr. At the end of the incubation membranes were treated as described above to separate free ligand from bound. The amount of [t2q]-D-AIa6-GnRH-EA specifically bound was compared with a standard curve generated by incubating various concentrations of a bovine pituitary membrane pool (.86 to 6.82 pmol GnRH receptor) with [t2q] D.Ala 6. GnRH-EA as described previously. The concentration of receptors for GnRH present in the pooled bovine pituitary homogenate was determined by Scatchard analysis (15).
Analys is o f H o r m o n e Conten t . Anterior pituitary content of IM (16) and FSH (1) were determined by radioimmunoassay. Hypothalamic content of GnRI-I was determined by radioimmunoassay (17) following extraction from POA and HYP with methanol-formic acid (9:1).
Analysis o f Data. Data were subjected to analysis of variance, and differences among treatment means were determined by Student-Neuman Keuls multiple range test (18).
RESULTS AND DISCUSSION
Val idat ion o f Estradiol-Receptor Assay. Binding of 3H-estradiol to bovine pituitary cytosol at 4C reaches equilibrium by 16 hr and is maintained through at least 24 hr of incubation (Figure 1). Thus, in all subsequent experiments, the incubation time was 20 hr.
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TIME OF INCUBATION (hr)
Fig. 1. T ime course for b ind ing o f 5H-estradiol to cytosol f rom the bovine anter ior pi tui tary. M a x i m u m b ind ing occu r red at 20 h r o f incuba t ion at 4C.
126 NETT, CERMAK, BRADEN, MANNS AND NISWENDER
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Fig. 2. Saturation and Scatchard (inset) analyses of the specific binding of 3H-estradiol by cytosol from bovine anterior pituitary.
Solubi l ized bov ine pi tu i tary recep to rs for estradiol were saturated by 3.33 nM 3H-estradiol wi th a K~ of 2.97 X 109 M -1 (Figure 2). In subsequen t saturat ion assays, 5 nM 3H-estradiol was used. The relat ive affinity of the es t rad io l - receptor for var ious s teroids is l is ted in Table 1. The r ecep to r b o u n d estrogens relat ive to thei r known b io logica l po tenc ies . Non-es t rogenic s teroids we re bound to a lesser degree.
V a l i d a t i o n o f GnRH-Receptor Assay. Binding of [~25I]-D-AIa6-GnRH-EA to bovine p i tu i ta ry m e m b r a n e at 4C reaches equ i l ib r ium wi th in 3 hr, and there was no decrease in r ecep to r h o r m o n e c o m p l e x at 4C for at least 12 hr. For de te rmina t ion of GnRH recep t o r content , incubat ions of [12q]-D-Ala6-GnRH-EA wi th individual p i tu i ta ry homogena te s we re for 12 hr.
The relat ive affinities of var ious agonists and antagonists of GnRH to bovine p i tu i ta ry m e m b r a n e are shown in Table 2. The abil i ty of each analog to inhibi t the b ind ing of [12q]-D-Ala6-GnRH-EA correla tes wel l wi th the i r r epor t ed bio- logical activity. Al though concen t ra t ions requ i red to d isplace [125I]-D-Ala6- GnRH-EA differed, the a m o u n t of total d i sp l acemen t by each analog was similar.
A s tandard curve and Scatchard p lo t used to quant i fy the n u m b e r of r ecep to rs for GnRH in each anter ior p i tu i ta ry are shown in Figures 3 and 4. The associat ion constant (K a) of the Scatchard p lo t was 3.55 X 10 *0 M -1. N u m b e r of recep tors (n) was .85 f m o l / m g fresh tissue.
Receptor Content. Conten t of r ecep to rs for GnRH in the anter ior p i tu i tary was u n c h a n g e d on days 6, 12 and 18 postes t rus (Figure 5). This was fo l lowed by a significant (P<.O01) decrease in r ecep to r n u m b e r on day 19 which con t inued th rough day 21. The only significant change observed in the n u m b e r of r ecep to r s for es t radiol was on day 18 w h e n con ten t of r ecep to rs increased more than two-fold over any o ther t ime obse rved (Figure 5).
TABLE 1. RELATIVE AFFINITY OF VARIOUS STEROIDS FOR BOVINE PITUITARY RECEPTORS FOR ESTRADIOL a
Fig. 3. Standard curve used to quantify membrane receptors for GnRH of individual bovine anterior pituitaries. Each point is the mean of triplicate determinations. The curve was constructed by measuring the binding of ~zSI-D-AIa~-GnRH-EA to increasing amounts of the anterior pituitary homogenate pool.
H o r m o n a l Content . No significant change was o b s e r v e d in the c o n t e n t of GnRH in either the POA or HYP during the estrous cycle (Figure 6). However, content of GnRH in the POA tended (P<. 1) to be lower on day 19 postestrus, the day receptors for GnRH in the anterior pituitary decreased. Mean content of GnRH was 3.0 -+ .5 ng in the POA and 6.5 _+ .7 ng in the medial basal hypothalamus.
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Fig. 4. Scatchard analysis of receptors for GnRH in a bovine anterior pituitary membrane pool.
128 NETT, CERMAK, BRADEN, MANNS AND NISWENDER
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ESTRADIOL
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DAY POSTESTRUS
Fig. 5. Content of receptors for GnRH (upper panel) and estradiol in the anterior pituitaries of cycling beef heifers at different times during the estrous cycle (Mean + SE, n=4 /group) . Upper panel: a,b bars with different superscripts differ (P<.001). Lower panel: a differs from c (P<.01); b differs from c (P<.05).
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Fig. 6. Content of GnRH (ng per hypothalamic area) in the POA and HYP of cycling beef heifers (Mean + SE, n=4 /g roup) .
HYPOTHALAMO-HYPOPHYSEAL AXIS IN BEEF COWS 129
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DAY POSTESTRUS
Fig. 7. Content of LH (upper panel) and FSH (lower panel) in the anterior pituitaries of cycling beef heifers (Mean + SE, n=4/group). Bars with different superscripts differ (P<.01).
Content of LH in the anter ior pi tui tary increased (P<.O1) from day 6 to day 18 (Figure 7). This was fo l lowed by a sharp decl ine on day 19 to a level lower (P< .05) than on any o ther day. By day 20 content of LH had re turned to a level comparable to that observed before day 19, and was maintained through day 21. Pituitary content of FSH was not different at any of the t imes observed (Figure 7).
C y c l e L e n g t h . It was not possible to obtain b lood samples f rom the cows used for these exper iments . However, it was de te rmined from three to six previous heat checks on each of the 24 cows used in this s tudy that their average cycle length was 19.8 -+ .4 day. This is consistent wi th all o ther data p resen ted suggesting the preovula tory surge of LH occur red day 19 postestrus (day of estrus = day 0).
DISCUSSION
A slight reduc t ion in content of GnRI-I in the POA on day 19 postestrns was the only significant change observed in hypothalamic tissue. Likewise, the most p r o n o u n c e d change in hypothalamic conten t of GnRH in ewes during the per iovula tory per iod was a decrease in the POA in conjunc t ion wi th the preovula tory surge of LH (10) . The median eminence is the pr imary site for storage of GnRI-I (19) , and one might exp ec t the most p r o n o u n c e d fluctuations in con ten t of GnRH as a result of a l tered release to be reflected at this locus. Unfortunately, the median eminence of a number of animals was damaged at
130 NETT, CERMAK, BRADEN, MANNS AND NISWENDER
the t ime of s laughter making accura te quant i f icat ion of GnRH from this tissue imposs ib le ; however , in sheep no change in GnRH conten t of the median e m i n e n c e was obse rved dur ing the pe r iovu la to ry pe r iod (10) .
That con ten t of LH in the anter ior p i tu i ta ry gland was decreased concomi tan t wi th r ecep to r s for GnRH in cows s laughtered on day 19 fur ther suggests the p reovu la to ry surge of LH ei ther had already occur red or was in the process of occur r ing in these animals (10 ,20 ,21 ) . Unfortunately, it was not poss ible to co l l ec t b lood samples pr ior to s laughter to confirm the occu r r ence of a surge of LH. On day 19, con ten t of LH in the anter ior p i tu i tary was significantly lower than at any o ther t ime obse rved dur ing the estrous cycle; however , this r educ t ion was t ransient re turn ing to a level comparab l e to that observed dur ing o ther stages of the estrous cycle by day 20. These data suggest that synthesis of LH by the anter ior p i tu i tary is capab le of rep len ish ing stores of LH wi th in 1 day. This is in contrast to our previous observat ions in sheep in wh ich p i tu i ta ry con ten t of LH had not re tu rned to pre-surge levels by 48 hr after the surge (10) . Likewise, Roche et al (20) r epor ted pi tu i tary conten t of LH to increase gradual ly t h roughou t the luteal phase of the cycle in ewes reaching a peak just p r io r to estrus. The con ten t of LH in cow pi tui tar ies repor ted by Schoenemann et al (3) was lower on the day of estrus and on day 7 of the cycle than on day 14. In the presen t study, p i tu i tary conten t of LH was c lear ly h igher on day 6 than on the p r e s u m e d day of estrus (day 19). The reason for the d i sc repant resul ts be t ween the e x p e r i m e n t r epor ted herein and that of Schoenemann et al (3) is not clear.
A reduc t ion in the n u m b e r of r ecep to rs for GnRH in the anter ior p i tu i tary occu r r ed on day 19 postestrus. This may have been due to a down-regula t ion of the n u m b e r of r ecep to rs for GnRH (10 ,14) as a resul t of an increase in the f r e q u e n c y of the GnRH pulses associated wi th the p reovu la to ry surge of LH. This is s u p p o r t e d by the fact that con ten t of GnRH in the POA tended to be lower on day 19. However , the n u m b e r of recep tors for GnRH in the anter ior p i tu i ta ry r ema ined low th rough at least day 21 but had re turned to normal by day 6 of the luteal phase. Similarly, Schoenemann et al (3) found that several days after the LH surge were requ i red before recep to rs for GnRH re turned to normal in heifers. The r educed recep to rs for GnRH may leave the p i tu i tary gland ref ractory to any fur ther s t imula t ion by GnRH even though LH stores we re rap id ly rep len ished . Synthesis of LH can occu r in the absence of GnRH s t imula t ion in ovine pi tu i tary cells main ta ined in v i t ro (22) .
An increase in the n u m b e r of cytosol ic r ecep to rs for estradiol in the anter ior p i tu i ta ry on day 18 postes t rus was the first change noted in the hypotha lamic- hypophysea l axis ur ing the pe r iovu la to ry pe r iod of the cow. The increase in recep to rs for estradiol may be act ing to al ter the sensit ivi ty of the anter ior p i tu i tary to c i rcu la t ing concent ra t ions of estradiol and may, a long wi th de- creasing se rum concen t ra t ions of p roges te rone , be respons ib le for the posi t ive f eedback effect of estradiol on the anter ior pitui tary. These findings are in contrast to those r epor t ed for the proes t rus rat (23) in wh ich a significant decrease in the n u m b e r of r ecep to rs for estradiol in p i tu i tary cytosol was observed. Previously, in the anter ior p i tu i ta ry gland of ewes no difference was obse rved b e t w e e n luteal and fol l icular phase concen t ra t ions of recep tors for estradiol (24) , bu t because samples we re co l l ec ted at only one t ime, it is poss ib le that a t ransient increase may not have been de tec ted .
From these data, it appears that the p reovu la to ry surge of LH in heifers is p r e c e d e d by an increase in the n u m b e r of r ecep to rs for estradiol in the pi tui tary
HYPOTHALAMO-HYPOPHYSEAL AXIS IN BEEF COWS 131
gland and is fo l lowed by a decrease in the number of receptors for GnRH. Pituitary stores of LH decreased concomit tan t ly wi th the number of receptors for GnRH. Thus, it appears that the hypophyseal changes that occur during the per iovulatory per iod of the cow are similar to those observed in other species.
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